Bottom Line:
Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized.Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs).Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

ABSTRACTEmpty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs). The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP or nuclear-targeted (n) LacZ) or secreted (human α1-antitrypsin, hA1AT) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ATL levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side-effects of rAAV8EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

fig5: Exacerbate liver transaminitis in BALB/c mice by increased ratios of empty adeno-associated virus (AAV) particles in rAAV8EGFP dosing formulations. Mouse sera were collected at different time points after vector perfusion. The serum alanine aminotransferase (ALT) levels were detected by ALT detection kits. The time courses of (a) the ALT levels and (b) comparison of ALT levels of different study groups are presented. Analysis of variance was used for comparing the experimental results with those from the groups with phosphate-buffered saline (PBS) or rAAV8 or AAV8 empty particles alone and was determined to be statistically significant. *P < 0.05, **P < 0.01 versus PBS; #P < 0.05, ##P < 0.01 versus rAAVEGFP alone. Finally, the groups of rAAV8EGFP vector mixed with different AAV8 empty particles were compared with the corresponding AAV8 empty particle alone, respectively, and the P value for each paired comparison is presented. rAAV, recombinant AAV.

Mentions:
On the other hand, our data also recapitulated previously reported side effects of rAAV transduction in BALB/c mice.15,20,21,23 First, although ALT elevations in the group treated with fully packaged rAAV8EGFP only were not as dramatic as what were previously reported,20–22 which may be attributed to possible differences in purification methods, serum ALT elevations were indeed observed as early as 1 week after vector injection, peaked at day 14 and not declined to those in the phosphate-buffered saline control group until the third week (Figure 5a). Second, infusions of 9 × 1011 CE and PE AAV8 particles to BALB/c mice all caused elevations of serum ALT levels in a pattern similar to those seen in 3 × 1011 GCs of fully packaged rAAV8EGFP only groups (Figure 5a), suggesting potential side effects of AAV8 capsids. Of note, addition of PE AAV8 particles derived from either rAAVEGFP or rAAVhA1AT production/purification process but not CE AAV8 particles at a REV of 75% to 3 × 1011 GCs of fully packaged rAAV8EGFP further aggravated ALT transaminitis, which was not resolved until 5 weeks after vector infusion (Figure 5a). A zoomed-in analysis of the ALT data at the 2-week time point, the peak of vector-caused transaminitis, confirmed that (i) administration of AAV particles, fully packaged, PE, and CE, all invoked slight but significant ALT elevations as compared with the phosphate-buffered saline group (all P < 0.01) and (ii) at a REV of 75%, exacerbation of vector-induced transaminitis was seen with empty AAV8 particles derived from rAAV8 production processes but not from the process without vector plasmid (Figure 5b). In other words, the empty capsids generated in the vector production/purification process appear to be more immunogenic than those produced in the absence of vector genome plasmid.

fig5: Exacerbate liver transaminitis in BALB/c mice by increased ratios of empty adeno-associated virus (AAV) particles in rAAV8EGFP dosing formulations. Mouse sera were collected at different time points after vector perfusion. The serum alanine aminotransferase (ALT) levels were detected by ALT detection kits. The time courses of (a) the ALT levels and (b) comparison of ALT levels of different study groups are presented. Analysis of variance was used for comparing the experimental results with those from the groups with phosphate-buffered saline (PBS) or rAAV8 or AAV8 empty particles alone and was determined to be statistically significant. *P < 0.05, **P < 0.01 versus PBS; #P < 0.05, ##P < 0.01 versus rAAVEGFP alone. Finally, the groups of rAAV8EGFP vector mixed with different AAV8 empty particles were compared with the corresponding AAV8 empty particle alone, respectively, and the P value for each paired comparison is presented. rAAV, recombinant AAV.

Mentions:
On the other hand, our data also recapitulated previously reported side effects of rAAV transduction in BALB/c mice.15,20,21,23 First, although ALT elevations in the group treated with fully packaged rAAV8EGFP only were not as dramatic as what were previously reported,20–22 which may be attributed to possible differences in purification methods, serum ALT elevations were indeed observed as early as 1 week after vector injection, peaked at day 14 and not declined to those in the phosphate-buffered saline control group until the third week (Figure 5a). Second, infusions of 9 × 1011 CE and PE AAV8 particles to BALB/c mice all caused elevations of serum ALT levels in a pattern similar to those seen in 3 × 1011 GCs of fully packaged rAAV8EGFP only groups (Figure 5a), suggesting potential side effects of AAV8 capsids. Of note, addition of PE AAV8 particles derived from either rAAVEGFP or rAAVhA1AT production/purification process but not CE AAV8 particles at a REV of 75% to 3 × 1011 GCs of fully packaged rAAV8EGFP further aggravated ALT transaminitis, which was not resolved until 5 weeks after vector infusion (Figure 5a). A zoomed-in analysis of the ALT data at the 2-week time point, the peak of vector-caused transaminitis, confirmed that (i) administration of AAV particles, fully packaged, PE, and CE, all invoked slight but significant ALT elevations as compared with the phosphate-buffered saline group (all P < 0.01) and (ii) at a REV of 75%, exacerbation of vector-induced transaminitis was seen with empty AAV8 particles derived from rAAV8 production processes but not from the process without vector plasmid (Figure 5b). In other words, the empty capsids generated in the vector production/purification process appear to be more immunogenic than those produced in the absence of vector genome plasmid.

Bottom Line:
Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized.Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs).Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

ABSTRACTEmpty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs). The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP or nuclear-targeted (n) LacZ) or secreted (human α1-antitrypsin, hA1AT) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ATL levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side-effects of rAAV8EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.